A rapid, efficient method for determining the presence of a wide range of microorganisms in food is needed by the food industry. Knowing the identities of most of the organisms in the microbial population in a food would be helpful in prediction of the shelf-life and for clarification of safety issues. The technology described will allow suppliers to characterize, monitor, and quantify microorganisms in ready-to-eat foods and will provide critical information concerning spoilage and contamination. We envision deployment of this technology for widespread use by major food manufacturers and testing agencies to provide cost-effective services. OBJECTIVES: High-throughput technology for identifying the presence of microorganisms that may lead to spoilage or illness would be of great value to the food industry. Such a system would reduce financial loss by allowing processors to design adequate early intervention measures including microbiological risk assessment and HACCP plans. The overall objective of this project is to demonstrate that our bacterial detection system can rapidly and effectively detect bacteria present in processed food items such as milk and vegetable salads without extensive cultivation. At the end of Phase I, we will deliver: 1) Universal PCR primers for discrimination of bacteria in food. 2) Procedures for efficiently extracting bacterial genomic DNA from food samples and reliable protocols for rapid gene amplification and sensitive MS analysis. 3) Procedures for preliminary quantification of bacteria detected. 4) A fully integrated system that generates robust, reproducible and interpretable results with actual food products. The research efforts on a pilot scale are designated as the four separate but interrelated tasks detailed in the Phase I work plan below. APPROACH: Task 1. Design six PCR primer pairs for simultaneous detection and discrimination of food related pathogenic and commensal bacteria. Task 2. Test each primer pair and optimize the detection of several major bacteria groups singly in a food matrix (milk or fruit juice) by spiking with known amounts of bacteria. Task 3. Test each primer pair and optimize the detection of a spiked mixture of five bacteria in a complex food matrix (vegetable salads). Task 4. Develop a plasmid-based DNA calibrant molecule for the quantification of bacteria in food samples. Demonstrate the ability to use this calibrant to quantify the presence of Pseudomonas in fluid milk products by the ESI-FTICR-MS coupled PCR system
